Diseases of the respiratory tract are among the most common diseases in humans and range in severity from being merely mild and annoying to life threatening. Examples of severe to life threatening respiratory diseases in humans include cystic fibrosis, asthma, emphysema, idiopathic pulmonary fibrosis and congenital deficiency of surfactant protein. Each of these diseases is suitable for gene therapy as a means of treatment provided that a gene delivery vehicle is available which delivers the appropriate gene in an effective and non-toxic manner. Gene therapy approaches which have been used or are contemplated for treatment of these respiratory diseases are reviewed in Canonico (1997, Gene Therapy for Chronic Inflammatory Diseases of the Lungs in Gene Therapy for Diseases of the Lung, K. L. Brigham, ed, Marcel Dekker, N.Y., pp.285-307).
Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians and, although the average life expectancy has increased to approximately 30 years in the past decade, there remains no effective cure for CF (Scanlin et aL, 1988, A. P. Fishman, ed. (McGraw-Hill, N.Y.) pp. 1273-1294; Welsh et al., 1995, Scriber et al, eds. (McGraw-Hill, N.Y.) pp. 3799-3876).
Patients having CF encode a mutated cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF is a multisystem disease, the most important and life threatening pathology occurs in the lung. Gene therapy has been proposed as a means of developing effective therapy to combat the pathology of CF. However, there are a plethora of problems associated with this approach, not the least of which is the lack of a suitable vehicle for delivery of the CFTR gene to humans.
Initial reports of gene therapy as a means of treating CF have focussed on airway epithelial cells as targets for the CFTR gene. Viral vectors have been used as vehicles for delivery of the CFTR gene to these cells in humans. However, the vectors themselves have proved to be sufficiently immunogenic so as to diminish any positive effect of the successful delivery of the CFTR gene to the affected cells of the individual (Wilson, 1995, J. Clin. Invest. 96:2547-2554; Crystal et al., 1995, Science 270:404-410).
Other vehicles which have been used as gene delivery vehicles include cationic lipids for transfer of genes to airway epithelial cells (Fasbender et al., 1995, Am. J. Physiol. 269:L45-L51). In addition, polylysines (poly-L-lysine) complexed with various glycoproteins, including transferrin targeted to the transferrin receptor, have been examined (Curiel et al., 1991, Proc. Natl. Acad. Sci. USA 88:8850-8854). Also reported, is the use of asialoglycoproteins targeted to hepatic cells through the asialoglycoprotein receptor (Wilson et al., 1992, J. Biol. Chem. 267:963-967), and the use of Tn antigen for gene transfer (Thurnher et al., 1994, Glycobiology 4:429-435). The above-referenced studies have largely been performed in cells other than airway epithelial cells. Moreover, complexes having glycoprotein as a component thereof are potentially immunogenic and therefore may not be of immediate value in human gene therapy.
Asthma is a disease of the industrialized 20th century, being described for the first time in the mid- 1800's. Exposure to otherwise harmless pollens and other allergens may set off a life threatening asthma attack in susceptible individuals, wherein constriction of the bronchioles renders a patient virtually unable to breathe. Asthma attacks are triggered by exposure to allergens which cause activated T lymphocytes of the T.sub.H 2 subset to secrete cytokines, primarily interleukin 4 (IL-4) and interleukin 5 (IL-5) setting off a cascade of events which ultimately leads to bronchioconstriction. IL-4 induces production by activated B lymphocytes of immunoglobulin (Ig) E which, in turn, induces the production of histamine from mast cells. IL-5 triggers the production by eosinophils of small fatty molecules known as leukotrienes. The combined action of histamine and leukotrienes causes blood vessels to leak and lung tissues to swell. The smooth muscles of the airways constrict and mucus production is induced which serves to further clog the already constricted airways.
Current asthma therapy is aimed at treating the end result, i.e., the airway constriction. However, targets other than the end point may be more amenable to therapy, particularly gene therapy. In addition, asthma is believed to have a genetic component, and in fact, the identification of an asthma gene has recently been announced (Vogel, 1997, Science 276:1327). This disease is therefore suitable for treatment using a gene therapy approach.
Alpha.sub.1 antitrypsin (.alpha.1AT) deficiency, like CF, is an inherited monogenic disorder having virtually no effective therapy beyond treatment for alleviation of the symptoms of the disease. .alpha.1AT deficiency is primarily associated with emphysema, a lung disease characterized by unopposed elastolytic destruction of the lung parenchyma. Although .alpha.1AT is synthesized primarily in liver cells, functional .alpha.1AT is responsible for over 95% of the antiprotease protection in the lower respiratory system. The most common genetic abnormality associated with premature emphysema is the Z allele. In this mutant allele, a lysine is substituted for glutamic acid at amino acid position 342 in .alpha.1AT, thereby altering the three dimensional configuration of the protein and affecting secretion of the protein from the cells in which it is synthesized. Other mutant alleles of the .alpha.1AT gene also contribute to the disease, and irrespective of the genetic abnormality, a critical threshold of an .alpha.1AT serum level of less than 10 .mu.M appears necessary for an individual to develop pulmonary emphysema.
Both the liver and the lung have been targeted for gene therapy as a means of treating .alpha.1AT deficiency. With respect to the liver, although successful liver-directed .alpha.1AT gene therapy has been achieved using various strategies, serum .alpha.1AT levels in all of these systems were below what would be necessary for physiological correction of the deficiency. Adenovirus-mediated gene therapy directed to lung cells has been attempted. However, because of the problems associated with adenovirus-induced inflammation, this is not the preferred approach. The use of other viruses and of liposomes has also been contemplated as a means of delivering .alpha.1AT to lung cells (Canonico, supra).
Idiopathic pulmonary fibrosis (IPF) is a lethal disease with a median time from diagnosis to death of 3 to 5 years. Since, current therapies for IPF have marginal effect on improved lung function or overall survival, a gene therapy approach for treatment of this disease is justified. In IPF, an inflammatory response to an unidentified insult or injury occurs following an exuberant fibrotic response. The initial inflammatory response is predominantly neutrophilic but evolves to a predominant lymphocytic and monocytic response. As yet, no specific genetic defect has been identified; however, gene therapy targeted to specific sites in the disease pathway, has been contemplated. For example, antisense therapy targeting specific growth factors or cytokines implicated in IPF has been proposed, in addition to delivery of other genes such as the cyclo-oxygenase-2 gene, the latter of which may block the effects of certain proinflammatory cytokines (Canonico, supra).
Congenital deficiency of surfactant protein results in severe respiratory disease in infants. The fundamental importance of surfactant protein (SP)-B in pulmonary function has been elucidated from studies on infants unable to produce SP-B due a genetic defect which gives rise to a lethal neonatal respiratory disease. Respiratory failure in these infants was refractory to therapies which included mechanical ventilation, surfactant replacement and extracorporeal membrane oxygenation. A genetically based deficiency in production of a second surfactant protein, SP-C, may also contribute to the development of this disease. Since this disease is governed by genes which have been identified and in view of the absence of any effective current therapy for this disease, a gene therapy approach for treatment of SP-B and/or SP-C deficiency seems appropriate. For a discussion on congenital deficiency of surfactant protein, see Whitsett et al. (1995, Physiological Reviews 75:749-757) and Nogee et al. (1994, J. Clin. Invest. 93:1860-1863).
As an alternative to virus or lipid mediated gene therapy, it has been reported that substitution of polylysine with lactose residues facilitates a high level of transfection of HepG2 cells via galactose-specific membrane lectins (Midoux et al., 1993, Nucleic Acids Res. 21:871-878; Erbacher et al., 1995, Bioconj. Chem. 6:401-410). It is also known that partially gluconoylated polylysine is an efficient vehicle for reporter gene expression in a number of different cell types (Midoux et al. ,1995, International Application Publication No. WO 95/30020; U.S. Pat. No. 5,595,897).
Polylysine substituted with specific sugars such as mannose or fucose may be used to transfect human macrophages which have a membrane lectin for mannose and fucose (Erbacher et al, 1996, Hum. Gene Ther. 7:721-729). Further, complex asialo-oligosaccharides coupled to short polylysine polymers have been used to transfect DNA into HepG2 cells (Wadhwa et al., 1995, Bioconj. Chem. 6:283-291).
There remains an acute need for a suitable vehicle for delivery of genes to respiratory cells, which vehicle must be non-immunogenic. Given the paucity of information on the nature of endogenous lectins on human airway epithelial cells (Drickamer et al., 1993, Ann. Rev. Cell Biol. 9:237-264), the use of polylysine derivatized with specific carbohydrates for delivery of genes to airway epithelial cells could not be predicted to successfully facilitate introduction of genes into these cells.